The cytidine analogue gemcitabine is first line chemotherapy for the treatment of pancreatic cancer, and it has also shown promising results in the treatment of breast cancer and non-small cell lung cancer. Gemcitabine has its effect as a result of a "pathway" that includes drug transporters, enzymes catalyzing drug activation and inactivation, and drug targets. However, very little is known with regard to determinants of variation in gemcitabine response, especially single nucleotide polymorphisms (SNPs) in genes outside of the pathway described by our current knowledge of this drug. In order to identify additional genes of importance for variation in gemcitabine response, we have used 300 Human Variation Panel lymphoblastoid cell line as a model system for common genetic variation to perform genome-wide expression association studies to identify genes with expression levels that were significantly associated with variation in gemcitabine cytotoxicity (IC50 values). One top candidate gene, FKBP5, a gene encoding a 51 kDa immunophilin, was shown to affect the apoptotic pathway in response to gemcitabine. Specifically, lower expression of FKBP5 was associated with resistance to gemcitabine-induced cytotoxicity. We also demonstrated an inhibitory role for FKBP5 in AKT phosphorylation. As a result, we hypothesize that FKBP5 affects gemcitabine response by negatively regulating AKT activation and that genetic variation associated with FKBP5 gene expression and protein function might contribute significantly to variation in gemcitabine response. In this application, we propose to determine mechanisms by which FKBP5 regulates AKT activation, followed by testing the role of FKBP5 in gemcitabine response using mice models and tumor samples from pancreatic cancer patients treated with gemcitabine. In addition, we will also determine gene sequence variation that is associated with FKBP5 gene expression and response to gemcitabine using 300 lymphoblastoid cell lines, followed by performing functional genomic studies with these SNPs. Finally, we will perform a genotype-phenotype correlation study with DNA from pancreatic cancer patients to determine whether SNPs that affect FKBP5 expression and/or protein function might influence response to gemcitabine when used to treat pancreatic cancer. In summary, this comprehensive series of experiments will enhance our understanding of mechanisms of gemcitabine resistance and may identify biomarkers that might help predict gemcitabine response in the treatment of pancreatic cancer. PUBLIC HEALTH RELEVANCE: The cytidine analogue gemcitabine is first line chemotherapy for the treatment of pancreatic cancer. However, very little is known with regard to determinants of variation in gemcitabine response, especially single nucleotide polymorphisms (SNPs) in genes outside of the "pathway" described by our current knowledge of the metabolism and "targets" for this drug. In order to identify additional genes of importance for variation in gemcitabine response, we have used 300 Human Variation Panel lymphoblastoid cell lines as a model system, together with genome-wide approaches to identify one top candidate gene, FKBP5, for which expression was significantly associated with gemcitabine sensitivity. In this application, based on extensive preliminary data, we propose to investigate mechanisms by which FKBP5 regulates response to gemcitabine and to identify genetic variation in FKBP5 that might be used as a biomarker to help predict gemcitabine response in the treatment of pancreatic cancer.